This invention pertains to the transformation, on the one hand, and organogenic regeneration, on the other hand, of cotton plants and kenaf plants, both commercially important crops. While the overall system is applicable to both crops, which are members of the same family, cotton transformation and regeneration is discussed first, followed by kenaf regeneration. Both nuclear and plastid transformation are embraced.
Cotton
Cotton has been traditionally recalcitrant to regeneration in vitro. Most regeneration successes entailed the sole use of Coker lines which respond in tissue culture but are not agronomically important (Chlan et al., 1995; Firoozabady et al., 1987; Peeters et al., 1994; Shoemaker et al., 1986; Umbeck et al. 1987). Most (if not all) developed regeneration protocols entail the production of embryogenic callus from seedling explants such as cotyledon and hypocotyl sections, followed by the formation of somatic embryos with subsequent germination and conversion into mature cotton plants (Firoozabady and DeBoer, 1993; Firoozabady et al., 1987; Peeters et al., 1994; Rajasekaran et al., 1996; Shoemaker et al., 1986; Umbeck et al. 1987; U.S. Pat. Nos. 5,159,135, 5,244,802). This type of regeneration procedure could take up to 40 weeks and could produce unwanted mutations due to the presence of a prolonged callus phase prior to regeneration.
Cotton tissues have been successfully transformed with A. tumefaciens prior to generation of embryogenic callus used in regeneration (Firoozabady et al., 1987; Rajasekaran et al., 1996; Umbeck et al. 1987). Cotton has also been transformed via biolistics with tissues also undergoing regeneration via somatic embryogenesis (Rajasekaran et al., 1996). A few protocols have recently utilized intact meristem-tips as targets in biolistics-based transformations with regeneration occurring via a more direct organogenic route (Chlan et al., 1995; Finer and McMullen, 1990; McCabe and Martinelli, 1993). Although this has overcome some regeneration obstacles, it is technically demanding. Due to their extremely small size (&lt;1.0 mm), the meristem-tips have to be excised with the aid of a dissecting microscope and once isolated, need to be utilized shortly thereafter.
Kenaf Kenaf (Hibiscus cannabinus L.), is a herbaceous annual fiber crop in the Malvaceae family. Since kenaf can be cultivated in a wide range of soils and climatic conditions, and requires only modest labor input, it is generally less expensive to produce when compared to most other fiber crops (Dempsey, 1975). Kenaf has been cultivated for use in various products, which include woven and non-woven textiles, newsprint, animal bedding, and potting soil mixes (Goforth and Fuller, 1994). Considerable interest has been shown for use of kenaf as a source of pulp for papermaking as newsprint made from kenaf is of high quality displaying high degree of smoothness and printability (Sij, 1987). The paper also has excellent ink-retention characteristics and its strength is well-suited to high speed newspaper presses.
Various pathogens adversely affect kenaf growth in the United States. Root-knot nematodes [Meloidogyne incognita (Kofoid and White) Chitwood] can contribute to yield losses up to 50% (Lawrence and McLean, 1991). In addition, Cristulariella moricola Hino (same as Cristulariella pyramidalis Waterman and Marshall) attacks kenaf leaves contributing to premature leaf abscission (Neely and Evers, 1976). In fact, this fungal pathogen has become a serious problem in the southern United States in recent years. The fungus causes a bull's-eye or zonate leafspot on both woody and annual plants, including 73 species in 36 families throughout the south central and south eastern United States. When kenaf was infected by the fungus, symptoms first appeared as necrotic lesions on leaves on the lower one-third of the plants (Neely and Evers, 1976). In 14 days, 50-75% of the leaves were found to be affected, at which time they usually abscised. By the time kenaf was ready for harvest, approximately 75% of the plant was defoliated (Neely and Evers, 1976). Cultivars from the following breeding series: Cubano, Everglades, Guatamela, and Tainung were all found to be susceptible to this fungal disease (B. S. Baldwin, personal communication).
Since all known kenaf cultivars are susceptible to C. moricola, there appeared to be little resistance available in the kenaf genome. Therefor, in vitro selection for resistant transformants would be an alternate way of establishing C. moricola resistant kenaf.
Accordingly, it remains an object of those of skill in the art to develop a method for regeneration of cotton and kenaf, in vitro, with an eye to obtaining genetic variation providing desirable qualities. In particular, it is a further object of those of skill in the art to obtain a method for transforming plant tissues with exogenous DNA, or obtaining mutation of endogenous DNA, and regenerating the tissues containing these DNA alterations and additions into mature, fertile plants.